C2 domains from different Ca2+ signaling pathways display functional and mechanistic diversity

The ubiquitous C2 domain is a conserved Ca2+ triggered membrane-docking module that targets numerous signaling proteins to membrane surfaces where they regulate diverse processes critical for cell signaling. In this study, we quantitatively compared the equilibrium and kinetic parameters of C2 domains isolated from three functionally distinct signaling proteins: cytosolic phospholipase A2-alpha (cPLA2-alpha), protein kinase C-beta (PKC-beta), and synaptotagmin-IA (Syt-IA). The results show that equilibrium C2 domain docking to mixed phosphatidylcholine and phosphatidylserine membranes occurs at micromolar Ca2+ concentrations for the cPLA2-alpha C2 domain, but requires 3- and 10-fold higher Ca2+ concentrations for the PKC-beta and Syt-IA C2 domains ([Ca2+](1/2) = 4.7, 16, 48 microM, respectively). The Ca2+ triggered membrane docking reaction proceeds in at least two steps: rapid Ca2+ binding followed by slow membrane association. The greater Ca2+ sensitivity of the cPLA2-alpha domain results from its higher intrinsic Ca2+ affinity in the first step compared to the other domains. Assembly and disassembly of the ternary complex in response to rapid Ca2+ addition and removal, respectively, require greater than 400 ms for the cPLA2-alpha domain, compared to 13 ms for the PKC-beta domain and only 6 ms for the Syt-IA domain. Docking of the cPLA2-alpha domain to zwitterionic lipids is triggered by the binding of two Ca2+ ions and is stabilized via hydrophobic interactions, whereas docking of either the PKC-beta or the Syt-IA domain to anionic lipids is triggered by at least three Ca2+ ions and is maintained by electrostatic interactions. Thus, despite their sequence and architectural similarity, C2 domains are functionally specialized modules exhibiting equilibrium and kinetic parameters optimized for distinct Ca2+ signaling applications. This specialization is provided by the carefully tuned structural and electrostatic parameters of their Ca2+ and membrane-binding loops, which yield distinct patterns of Ca2+ coordination and contrasting mechanisms of membrane docking.